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Biphasic Effects of Doxorubicin on the Calcium Release Channel From Sarcoplasmic Reticulum of Cardiac Muscle Karol Ondrias, Louis Borgatta, Do Han Kim, and Barbara E. Ehrlich To define the mechanism of doxorubicin cardiotoxicity, the effects of doxorubicin and caffeine examined on calcium release channels from cardiac sarcoplasmic reticulum. We found that calcium release from cardiac sarcoplasmic reticulum vesicles was induced by both compounds. When sarcoplasmic reticulum vesicles were incorporated into planar lipid bilayers, calcium-permeable channels were observed. Addition of caffeine (2.5-10 mM) increased channel open probability from less than 0.1% to 40%, and this effect persisted for a mean of 44 minutes. In contrast, doxorubicin (2.5-10 ,uM) had a biphasic effect; initially, doxorubicin activated the channel, whereas after a mean of 8 minutes, the channel became irreversibly inhibited. Although the degree of channel activation by doxorubicin was concentration dependent, the time needed to inactivate the channel was concentration independent. Pretreatment with dithiothreitol (0.2 mM) prevented doxorubicin-induced channel inactivation, and channel activity persisted for an average of 58 minutes. Dithiothreitol alone did not alter channel open probability. Our results support the hypotheses that 1) the integrity of sulfhydryl groups is important for some aspects of calcium release channel function and 2) activation and inactivation of the channel are separable processes. The biphasic effect of doxorubicin on channel function may also correspond to the clinically observed adverse effects of doxorubicin, a widely used chemotherapeutic agent that, after prolonged usage, causes a dilated cardiomyopathy. (Circulation Research 1990;67:1167-1174)
were
C alcium stored in the sarcoplasmic reticulum (SR) of cardiac or skeletal muscle is mobilized to initiate a contraction by activating ion channels called calcium release channels. These channels, which are concentrated in the T tubule/SR junction of skeletal muscle,1-3 can be purifiedl,3 and reconstituted.3 In addition, the channel protein has recently been cloned.4 Comparisons of biochemical and molecular properties of the channels from cardiac and skeletal muscle have shown that these channels are similar.5-7 However, the physiological trigger for opening the channel appears to be different in the two types of striated muscle,8'9 and differences in some of the functional properties of the channels from the two types of muscle have been From the Departments of Medicine (K.O., L.B., D.H.K., B.E.E.) and Physiology (B.E.E.), University of Connecticut, Farmington, Conn. Supported by National Institutes of Health grant HL-33026 and a grant-in-aid from the American Heart Association, Connecticut Affiliate. D.H.K. is an Established Investigator of the American Heart Association. B.E.E. is a Pew Scholar in the Biomedical Sciences. Address for correspondence: Dr. Barbara E. Ehrlich, Division of Cardiology, University of Connecticut, Farmington, CT 06030. Received February 6, 1990; accepted July 2, 1990.
described (compare Smith et al5 and Rousseau et
a16). Calcium release from SR vesicles made from both muscles can be initiated by a number of compounds, although different sensitivities to the compounds have been reported.'0 Calcium and ATP appear to be important for the activity of the calcium release channel under physiological conditions, particularly in cardiac muscle, where the trigger for excitationcontraction coupling is thought to be calcium from the extracellular solution.9 Other compounds such as caffeine, halothane, and doxorubicin have effects on calcium release from vesicles, although this activation may be related to the adverse effects of the compound. Doxorubicin (Adriamycin, Adria Laboratories, Dublin, Ohio), a widely used chemotherapeutic agent, activates calcium release from SR vesicles and has been used as a photoaffinity label for the calcium release channel complex.1' Clinically, this compound is cardiotoxic.12-14 It has been hypothesized that the doxorubicin-induced cardiotoxicity is related to lipid peroxidation, oxidation of critical sulfhydryl groups, and/or triggering of calcium efflux from SR.10,15-18 These effects may be interrelated. For example,
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oxidation of sulfhydryl groups on the calcium release channel of SR may alter the activity of the channel. The experiments in this report show that doxorubicin exerts a biphasic effect on the calcium release channel of cardiac SR. The initial effect of doxorubicin is to activate the channel, whereas prolonged exposure irreversibly inhibits the channel. This contrasts with the action of caffeine, which produces a prolonged activation of the calcium release channel. In addition, we found that it was possible to "protect" the channel from doxorubicin-induced inhibition by pretreating the channels with dithiothreitol (DTT), an agent that maintains sulfhydryl groups in the reduced form.
Materials and Methods SR vesicles prepared from canine heart as described previously19 were suspended in a buffer containing 150 mM KCl, 20 mM 3(N-morpholino)propanesulfonic acid (MOPS) (pH 6.8), 0.1 mM phenylmethylsulfonyl fluoride, 10 mg/I aprotinin, 0.8 mg/l antipain, 2 mg/l trypsin inhibitor, and 0.3 M sucrose10,20 and quickly frozen in liquid nitrogen and kept at -80° C. Vesicles were used within 4 weeks after preparation. Protein concentrations were determined by the Lowry method,21 using bovine serum albumin as a standard. Calcium release was monitored using 49Ca2 as described previously.22 Briefly, the SR vesicles (0.05 mg/ml) were incubated at 370 C for 5 minutes in a reaction mixture containing 0.15 M KCI, 20 mM MOPS (pH 6.8), 5 mM MgCl2, 10 mM NaN3, 0.26 mM EGTA, and 0.065 mM 45CaCl2 (104 cpm/nmol). Active calcium uptake was started by addition of 2 mM ATP to the reaction mixture. At a steady state of calcium loading, calcium release was initiated by addition of doxorubicin or caffeine and stopped at various times by filtration of aliquots of the reaction mixture with 0.45-,um filters (Millipore Corp., Bedford, Mass.). After filtration, each filter was immediately washed twice with 2.5 ml of 0.15 M KCl, 20 mM MOPS (pH 6.8), 30 mM EGTA, and 15 gM ruthenium red.22 The filters were then dried, and their radioactivity was counted. SR vesicles also were incorporated into preformed planar lipid bilayers that were formed by painting a lipid/decane solution across a hole in a Teflon partition that separated two lucite compartments.23 Channel incorporation was accomplished using the method outlined by Smith et al.5 Briefly, vesicles were added to one compartment after the membrane was formed, usually the cis compartment. Incorporation was monitored in the presence of a chloride and an osmotic gradient across the membrane [cis solution: 600 mM N-methyl-D-glucamine chloride, 20 mM HEPES, 10 mM CaCl2, 0.2 mM EGTA, pH 7.3; trans solution: 250 mM HEPES, 53 mM Ca(OH)2, pH 7.3]. The insertion of a chloride-permeable channel was the signal that a vesicle had fused with the bilayer. These channels are convenient markers because they are present in SR membranes, they are
large conductance channels, and they are open most of the time. To monitor calcium release channels, the bilayer chamber was then perfused with a chloridefree solution: 250 mM HEPES-Tris, 1 mM EGTA, and 0.5 mM CaCl2 (0.1 ,uM free calcium), pH 7.3. In addition, the chloride-free solution was isosmotic to the trans solution to prevent incorporation of additional vesicles into the bilayer. Channel insertion and subsequent experiments were monitored under voltage-clamp conditions with a pair of Ag/AgCI electrodes contacting the solutions via CsCl junctions. The channel currents were amplified using a patch-clamp amplifier (Yale model MK5, Warner Instruments, Hamden, Conn., or Axopatch Tb, Axon Instruments, Burlingame, Calif.) and recorded on chart (General Scanning, Watertown, Mass.) and tape (Dagan Corp, Minneapolis, Minn.) recorders. Data were analyzed after filtering with an eight-pole Bessel filter (Frequency Devices, Haverhill, Mass.) to 300 Hz and digitizing at 1 kHz to transfer to a PDP 11/73 (Indec Systems, Sunnyvale,
Calif). Doxorubicin and caffeine were purchased from Sigma, St. Louis. Lipids were purchased from Avanti Polar Lipids, Birmingham, Ala. All other reagents used were of analytical grade. Results Doxorubicin or Caffeine Induces Rapid Calcium Release From Cardiac SR Vesicles Caffeine and doxorubicin initiated release of calcium from cardiac SR vesicles that had been loaded with 45Ca2 (-15 nmol/mg SR) in the presence of 2 mM ATP. At 7 minutes after addition of ATP, when the calcium loading had reached a steady state, addition of 2 mM caffeine caused approximately 30% of the loaded calcium to be released from the SR within 10 seconds (Figure 1, left panel), while 40% of the loaded calcium was released by addition of 25 gM doxorubicin (Figure 1, right panel). Further increase in the drug concentrations did not change the amount of calcium released. Similar results have been obtained with caffeine and doxorubicin when calcium efflux from cardiac SR vesicles was monitored with the calcium indicator antipyrylazo III (Reference 24 and data not shown).
Doxorubicin or Caffeine Opens Calcium Release Channels From Cardiac SR Incorporated Into Planar Lipid Bilayers To show that caffeine- and doxorubicin-induced calcium release from SR vesicles was channel-mediated, SR vesicles from cardiac muscle were incorporated into planar lipid bilayers and channel currents were recorded. Channel activity in the presence of 0.1 ,uM free calcium is shown in Figure 2 (top two tracings of each panel). In the absence of drugs, the probability of finding the channel open under these conditions was generally less than 1%. Addition of either caffeine (Figure 2, bottom two tracings of left
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Ondrias et al Biphasic Modulation of SR Calcium Channels by Doxorubicin 20.
20.
f f~~~~~~~~ 25 pM Doxorublcin
2 mM Caffin
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FIGURE 1. Graphs showing time course of calcium uptake and release triggered by caffeine or doxorubicin from cardiac sarcoplasmic reticulum (SR) vesicles. Calcium uptake was started by the addition of 2 mMATP to the reaction mixture. Release was triggered by addition of 2 mM caffeine (left panel) or 25 MM doxorubicin (right panel) and terminated by filtration.
panel) or doxorubicin (Figure 2, bottom two tracings of right panel) increased the open probability to 5-90%, depending on the concentration of the agent. In no case did the addition of these compounds fail to activate a channel, and in some cases, especially with caffeine, two or three channels became active. The responses of the channels to caffeine and doxorubicin were similar, and the single-channel conductance of the channel was the same after activation of the channel with calcium, caffeine, or doxorubicin (Figure 3). The current-voltage relations of the singlechannel currents, plotted for all three activating agents, yielded similar slopes, which determine the single-channel conductance. The slope conductance (at 0 mV) was 100 pS for all three agents. Caffeine-Induced Opening of Calcium Release Channels Is Maintained Addition of caffeine to the cytoplasmic side of the channel increased channel activity, and in eight of 15 experiments, caffeine opened more than one channel. In the control period of the experiment (Figure 4, top plot of upper left panel) the channel was open
less than 0.1%. Only a 2-minute control segment is shown, but this behavior was maintained for 10 minutes. Addition of 2.5 mM caffeine at the beginning of the segment shown led to increased channel activity (Figure 4, second plot of upper left panel). The third and fourth plots show channel activity 10 and 20 minutes after caffeine addition. Although the mean open time varied between 52% and 29% in this experiment, it does not indicate that the activity is decreasing with time. Variation in the mean activity level as seen in this figure was found in virtually all experiments performed, regardless of the concentration of caffeine used. This variability appears to be a property of the calcium release channel from SR. For example, two additional experiments are shown in Figure 5, in which the degree of activation varied between 18% and 82%. The duration of activation was similar after addition of either 2.5 or 5 mM caffeine (Figure 5). In 10 experiments in which caffeine was the activator, the channels were active for a period greater than 15 minutes (16-90 minutes; mean, 44 minutes). A second addition of caffeine reactivated the channel.
Control
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FIGURE 2. Recordings showing acute effects of caffeine and doxorubicin on single-channel currents of cardiac calcium release channels. Single-channel recordings in control conditions (0.1 pum free calcium) had a open probability of 200 times less intense in the ultraviolet range). In summary, we have shown that both caffeine and doxorubicin can activate the calcium release channel from cardiac SR but that doxorubicin induced inhibition of the channel after prolonged exposure. Dox-
1173
orubicin-induced inactivation of the channel was prevented by pretreating the system with DPI?. These experiments show that activation and inactivation of the calcium release channel can be separated, suggesting that different biochemical mechanisms are involved in the two responses to doxorubicin addition. These experiments also show that the biphasic effects of doxorubicin on the calcium release channel can be related to the observed cardiotoxicity. Acknowledgments We gratefully acknowledge Dr. James Watras for assistance in data analysis and Drs. Arnold Katz and James Watras, Department of Medicine, University of Connecticut, Farmington, for helpful comments on the manuscript. References 1. Imagawa T, Smith, JS, Coronado R, Campell KP: Purified ryanodine receptor from skeletal muscle sarcoplasmic reticulum is the calcium permeable pore of the calcium release
channel. J Biol Chem 1987;262:16636-16643 2. Inui M, Saito A, Fleischer S: Isolation of the ryanodine receptor from cardiac sarcoplasmic reticulum and identity with the feet structures. J Biol Chem 1987;262:15637-15642 3. Lai FA, Erickson HP, Rousseau E, Liu Q-Y, Meissner G: Purification and reconstitution of the calcium release channel from skeletal muscle. Nature 1988;331:315-319 4. Takeshima H, Nishimura S, Matsumoto T, Ishida H, Kangawa K, Minamino N, Matsuo H, Ueda M, Hanaoka M, Hirose T, Numa S: Primary structure and expression from complementary DNA of skeletal muscle ryanodine receptor. Nature 1989;339:439-445 5. Smith J, Coronado R, Meissner G: Single channel measurements of the calcium release channel from skeletal muscle sarcoplasmic reticulum: Activation by calcium and ATP and modulation by magnesium. J Gen Physiol 1986;88:573-588 6. Rousseau E, Smith J, Henderson J, Meissner G: Single channel and calcium flux measurements of the cardiac sarcoplasmic reticulum calcium channel. Biophys J 1986;50: 1009-1014 7. Ehrlich BE, Watras J: Inositol 1,4,5-trisphosphate activates a channel from smooth muscle sarcoplasmic reticulum. Nature 1988;336:583-586 8. Tanabe T, Beam KG, Powell JA, Numa S: Restoration of excitation-contraction coupling and slow calcium current in dysgenic muscle by dihydropyridine receptor complementary DNA. Nature 1988;336:134-149 9. Fabiato A, Fabiato F: Use of chlorotetracycline fluorescence to demonstrate calcium-induced release of calcium from the
10. 11. 12.
13. 14.
sarcoplasmic reticulum of the skinned cardiac cells. Nature 1979;281:146-148 Kim DH, Landry AB III, Lee YS, Katz AM: Doxorubicininduced calcium release from cardiac sarcoplasmic reticulum vesicles. J Mol Cell Cardiol 1989;21:433-436 Zorzato F, Margreth A, Volpe P: Direct photoaffinity labeling of junctional sarcoplasmic reticulum with [14Cjdoxorubicin. J Biol Chem 1986;261:13252-13257 Arena E, D'Alessandro N, Dusonchet L, Gebbia L, Gerbasi F, Sanguedolce R, Rausa L: Influence of pharmacokinetic variations on the pharmacologic properties of adriamycin, in Carter SK, DiMarco A, Ghione M, Krakoff IH, Mathe G (eds): International Symposium on Adriamycin. Berlin/ Heidelberg/New York, Springer-Verlag, 1972, pp 9-22 Buja LM, Ferrans VJ, Mayer RJ, Roberts WC, Henderson ES: Cardiac ultrastructural changes induced by daunorubicin therapy. Cancer 1973;32:771-778 Lefrak EA, Pitha J, Rosenheim S, Gottlieb J: A clinicopathologic analysis of adriamycin cardiotoxicity. Cancer 1973;32: 302-314
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15. Abramson JJ, Buck E, Salama G, Casida JE, Pessah IN: Mechanism of anthraquinone-induced calcium release from skeletal muscle sarcoplasmic reticulum. J Biol Chem 1988;263: 18750-18758 16. Powis G: Free radical formation by antitumor quinones. Free Radic Biol Med 1989;6:63-101 17. Singal PK: Subcellular effects of adriamycin in the heart: A concise review. J Mol Cell Cardiol 1987;9:817-828 18. Zorzato F, Salviati G, Facchinetti T, Volpe P: Doxorubicin induces calcium release from terminal cisternae of skeletal muscle. J Biol Chem 1985;260:7349-7355 19. Kranias EG, Schwartz A, Jungmann RA: Characterization of cyclic 3'5 '-AMP-dependent protein kinase in sarcoplasmic reticulum and cytosol of canine myocardium. Biochim Biophys Acta 1982;709:28-37 20. Kim DH, Ikemoto N: Involvement of 60-kilodalton phosphoprotein in the regulation of calcium release from skeletal muscle sarcoplasmic reticulum. J Biol Chem 1986;261: 11674-11679 21. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265-275 22. Kim HW, Kim DH, Ikemoto N, Kranias EG: Lack of effect of calcium calmodulin-dependent phosphorylation on Ca2' release from cardiac sarcoplasmic reticulum. Biochim Biophys Acta 1987;903:333-340 23. Mueller P, Rudin DO, Tien HT, Wescott WC: Methods for the incorporation of single bimolecular lipid membranes in aqueous solutions. J Phys Chem 1963;67:534-535 24. Watras J, Benevolensky D: Inositol 1,4,5, triphosphate-induced calcium release from canine aortic sarcoplasmic reticulum vesicles. Biochim Biophys Acta 1987;931:354-363 25. Kim DH, Ohnishi ST, Ikemoto N: Kinetic studies of calcium release from sarcoplasmic reticulum in vitro. J Biol Chem 1983;258:9662-9668
26. Benevolensky DS, Menshikova EV, Watras J, Levitsky DO, Ritov VB: Characterization of Ca2+ release from the sarcoplasmic reticulum of myocardium and vascular smooth muscle. Biomed Biochim Acta 1987;46:S393-S398 27. Rousseau E, LaDine J, Liu Q-Y, Meissner G: Activation of the calcium release channel of the skeletal muscle sarcoplasmic reticulum by caffeine and related compounds. Arch Biochem Biophys 1988;267:75-86 28. Rousseau E, Meissner G: Single cardiac sarcoplasmic reticulum Ca2+-release channel: Activation by caffeine. Am J Physiol 1989;256:H328-H333 29. Preisler HD, Gessner T, Azarnia N, Bolanowska W, Epstein J, Early AP, D'Arrigo P, Vogler R, Winton L, Chervenik P, Joyce R, Lee H, Steele R, Goldberg J, Gottlieb A, Browman G, Miller K, Grunwald H, Larson R, Brennan J: Relationship between plasma adriamycin levels and the outcome of remission induction therapy for acute nonlymphocytic leukemia. Cancer Chemother Pharmacol 1984;12:125-130 30. Cortes EP, Gupta M, Chou C, Amin VC, Folkers K: Adriamycin cardiotoxicity: Early detection by systolic time interval and possible prevention by coenzyme Q10. Cancer Treat Reports 1978;62:887-891 31. Harris RN, Doroshow JH: Effect of doxorubicin-enhanced hydrogen peroxide and hydroxyl radical formation on calcium sequestration by cardiac sarcoplasmic reticulum. Biochem Biophys Res Commun 1985;130:739-745 32. Singal PK, Pierce GN: Adriamycin stimulates low-affinity Ca2' binding and lipid peroxidation but depresses myocardial function. Am J Physiol 1986;250:H419-H425
KEY WORDS * sarcoplasmic reticulum * planar lipid bilayers doxorubicin cardiotoxicity * calcium channels * single ion channels
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Biphasic effects of doxorubicin on the calcium release channel from sarcoplasmic reticulum of cardiac muscle. K Ondrias, L Borgatta, D H Kim and B E Ehrlich Circ Res. 1990;67:1167-1174 doi: 10.1161/01.RES.67.5.1167 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1990 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571
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Biphasic Effects of Doxorubicin on the Calcium Release Channel From Sarcoplasmic Reticulum of Cardiac Muscle Karol Ondrias, Louis Borgatta, Do Han Kim, and Barbara E. Ehrlich To define the mechanism of doxorubicin cardiotoxicity, the effects of doxorubicin and caffeine examined on calcium release channels from cardiac sarcoplasmic reticulum. We found that calcium release from cardiac sarcoplasmic reticulum vesicles was induced by both compounds. When sarcoplasmic reticulum vesicles were incorporated into planar lipid bilayers, calcium-permeable channels were observed. Addition of caffeine (2.5-10 mM) increased channel open probability from less than 0.1% to 40%, and this effect persisted for a mean of 44 minutes. In contrast, doxorubicin (2.5-10 ,uM) had a biphasic effect; initially, doxorubicin activated the channel, whereas after a mean of 8 minutes, the channel became irreversibly inhibited. Although the degree of channel activation by doxorubicin was concentration dependent, the time needed to inactivate the channel was concentration independent. Pretreatment with dithiothreitol (0.2 mM) prevented doxorubicin-induced channel inactivation, and channel activity persisted for an average of 58 minutes. Dithiothreitol alone did not alter channel open probability. Our results support the hypotheses that 1) the integrity of sulfhydryl groups is important for some aspects of calcium release channel function and 2) activation and inactivation of the channel are separable processes. The biphasic effect of doxorubicin on channel function may also correspond to the clinically observed adverse effects of doxorubicin, a widely used chemotherapeutic agent that, after prolonged usage, causes a dilated cardiomyopathy. (Circulation Research 1990;67:1167-1174)
were
C alcium stored in the sarcoplasmic reticulum (SR) of cardiac or skeletal muscle is mobilized to initiate a contraction by activating ion channels called calcium release channels. These channels, which are concentrated in the T tubule/SR junction of skeletal muscle,1-3 can be purifiedl,3 and reconstituted.3 In addition, the channel protein has recently been cloned.4 Comparisons of biochemical and molecular properties of the channels from cardiac and skeletal muscle have shown that these channels are similar.5-7 However, the physiological trigger for opening the channel appears to be different in the two types of striated muscle,8'9 and differences in some of the functional properties of the channels from the two types of muscle have been From the Departments of Medicine (K.O., L.B., D.H.K., B.E.E.) and Physiology (B.E.E.), University of Connecticut, Farmington, Conn. Supported by National Institutes of Health grant HL-33026 and a grant-in-aid from the American Heart Association, Connecticut Affiliate. D.H.K. is an Established Investigator of the American Heart Association. B.E.E. is a Pew Scholar in the Biomedical Sciences. Address for correspondence: Dr. Barbara E. Ehrlich, Division of Cardiology, University of Connecticut, Farmington, CT 06030. Received February 6, 1990; accepted July 2, 1990.
described (compare Smith et al5 and Rousseau et
a16). Calcium release from SR vesicles made from both muscles can be initiated by a number of compounds, although different sensitivities to the compounds have been reported.'0 Calcium and ATP appear to be important for the activity of the calcium release channel under physiological conditions, particularly in cardiac muscle, where the trigger for excitationcontraction coupling is thought to be calcium from the extracellular solution.9 Other compounds such as caffeine, halothane, and doxorubicin have effects on calcium release from vesicles, although this activation may be related to the adverse effects of the compound. Doxorubicin (Adriamycin, Adria Laboratories, Dublin, Ohio), a widely used chemotherapeutic agent, activates calcium release from SR vesicles and has been used as a photoaffinity label for the calcium release channel complex.1' Clinically, this compound is cardiotoxic.12-14 It has been hypothesized that the doxorubicin-induced cardiotoxicity is related to lipid peroxidation, oxidation of critical sulfhydryl groups, and/or triggering of calcium efflux from SR.10,15-18 These effects may be interrelated. For example,
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oxidation of sulfhydryl groups on the calcium release channel of SR may alter the activity of the channel. The experiments in this report show that doxorubicin exerts a biphasic effect on the calcium release channel of cardiac SR. The initial effect of doxorubicin is to activate the channel, whereas prolonged exposure irreversibly inhibits the channel. This contrasts with the action of caffeine, which produces a prolonged activation of the calcium release channel. In addition, we found that it was possible to "protect" the channel from doxorubicin-induced inhibition by pretreating the channels with dithiothreitol (DTT), an agent that maintains sulfhydryl groups in the reduced form.
Materials and Methods SR vesicles prepared from canine heart as described previously19 were suspended in a buffer containing 150 mM KCl, 20 mM 3(N-morpholino)propanesulfonic acid (MOPS) (pH 6.8), 0.1 mM phenylmethylsulfonyl fluoride, 10 mg/I aprotinin, 0.8 mg/l antipain, 2 mg/l trypsin inhibitor, and 0.3 M sucrose10,20 and quickly frozen in liquid nitrogen and kept at -80° C. Vesicles were used within 4 weeks after preparation. Protein concentrations were determined by the Lowry method,21 using bovine serum albumin as a standard. Calcium release was monitored using 49Ca2 as described previously.22 Briefly, the SR vesicles (0.05 mg/ml) were incubated at 370 C for 5 minutes in a reaction mixture containing 0.15 M KCI, 20 mM MOPS (pH 6.8), 5 mM MgCl2, 10 mM NaN3, 0.26 mM EGTA, and 0.065 mM 45CaCl2 (104 cpm/nmol). Active calcium uptake was started by addition of 2 mM ATP to the reaction mixture. At a steady state of calcium loading, calcium release was initiated by addition of doxorubicin or caffeine and stopped at various times by filtration of aliquots of the reaction mixture with 0.45-,um filters (Millipore Corp., Bedford, Mass.). After filtration, each filter was immediately washed twice with 2.5 ml of 0.15 M KCl, 20 mM MOPS (pH 6.8), 30 mM EGTA, and 15 gM ruthenium red.22 The filters were then dried, and their radioactivity was counted. SR vesicles also were incorporated into preformed planar lipid bilayers that were formed by painting a lipid/decane solution across a hole in a Teflon partition that separated two lucite compartments.23 Channel incorporation was accomplished using the method outlined by Smith et al.5 Briefly, vesicles were added to one compartment after the membrane was formed, usually the cis compartment. Incorporation was monitored in the presence of a chloride and an osmotic gradient across the membrane [cis solution: 600 mM N-methyl-D-glucamine chloride, 20 mM HEPES, 10 mM CaCl2, 0.2 mM EGTA, pH 7.3; trans solution: 250 mM HEPES, 53 mM Ca(OH)2, pH 7.3]. The insertion of a chloride-permeable channel was the signal that a vesicle had fused with the bilayer. These channels are convenient markers because they are present in SR membranes, they are
large conductance channels, and they are open most of the time. To monitor calcium release channels, the bilayer chamber was then perfused with a chloridefree solution: 250 mM HEPES-Tris, 1 mM EGTA, and 0.5 mM CaCl2 (0.1 ,uM free calcium), pH 7.3. In addition, the chloride-free solution was isosmotic to the trans solution to prevent incorporation of additional vesicles into the bilayer. Channel insertion and subsequent experiments were monitored under voltage-clamp conditions with a pair of Ag/AgCI electrodes contacting the solutions via CsCl junctions. The channel currents were amplified using a patch-clamp amplifier (Yale model MK5, Warner Instruments, Hamden, Conn., or Axopatch Tb, Axon Instruments, Burlingame, Calif.) and recorded on chart (General Scanning, Watertown, Mass.) and tape (Dagan Corp, Minneapolis, Minn.) recorders. Data were analyzed after filtering with an eight-pole Bessel filter (Frequency Devices, Haverhill, Mass.) to 300 Hz and digitizing at 1 kHz to transfer to a PDP 11/73 (Indec Systems, Sunnyvale,
Calif). Doxorubicin and caffeine were purchased from Sigma, St. Louis. Lipids were purchased from Avanti Polar Lipids, Birmingham, Ala. All other reagents used were of analytical grade. Results Doxorubicin or Caffeine Induces Rapid Calcium Release From Cardiac SR Vesicles Caffeine and doxorubicin initiated release of calcium from cardiac SR vesicles that had been loaded with 45Ca2 (-15 nmol/mg SR) in the presence of 2 mM ATP. At 7 minutes after addition of ATP, when the calcium loading had reached a steady state, addition of 2 mM caffeine caused approximately 30% of the loaded calcium to be released from the SR within 10 seconds (Figure 1, left panel), while 40% of the loaded calcium was released by addition of 25 gM doxorubicin (Figure 1, right panel). Further increase in the drug concentrations did not change the amount of calcium released. Similar results have been obtained with caffeine and doxorubicin when calcium efflux from cardiac SR vesicles was monitored with the calcium indicator antipyrylazo III (Reference 24 and data not shown).
Doxorubicin or Caffeine Opens Calcium Release Channels From Cardiac SR Incorporated Into Planar Lipid Bilayers To show that caffeine- and doxorubicin-induced calcium release from SR vesicles was channel-mediated, SR vesicles from cardiac muscle were incorporated into planar lipid bilayers and channel currents were recorded. Channel activity in the presence of 0.1 ,uM free calcium is shown in Figure 2 (top two tracings of each panel). In the absence of drugs, the probability of finding the channel open under these conditions was generally less than 1%. Addition of either caffeine (Figure 2, bottom two tracings of left
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Ondrias et al Biphasic Modulation of SR Calcium Channels by Doxorubicin 20.
20.
f f~~~~~~~~ 25 pM Doxorublcin
2 mM Caffin
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/~~~~~~~~~~~~~~~~~~~~~
.5 E
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FIGURE 1. Graphs showing time course of calcium uptake and release triggered by caffeine or doxorubicin from cardiac sarcoplasmic reticulum (SR) vesicles. Calcium uptake was started by the addition of 2 mMATP to the reaction mixture. Release was triggered by addition of 2 mM caffeine (left panel) or 25 MM doxorubicin (right panel) and terminated by filtration.
panel) or doxorubicin (Figure 2, bottom two tracings of right panel) increased the open probability to 5-90%, depending on the concentration of the agent. In no case did the addition of these compounds fail to activate a channel, and in some cases, especially with caffeine, two or three channels became active. The responses of the channels to caffeine and doxorubicin were similar, and the single-channel conductance of the channel was the same after activation of the channel with calcium, caffeine, or doxorubicin (Figure 3). The current-voltage relations of the singlechannel currents, plotted for all three activating agents, yielded similar slopes, which determine the single-channel conductance. The slope conductance (at 0 mV) was 100 pS for all three agents. Caffeine-Induced Opening of Calcium Release Channels Is Maintained Addition of caffeine to the cytoplasmic side of the channel increased channel activity, and in eight of 15 experiments, caffeine opened more than one channel. In the control period of the experiment (Figure 4, top plot of upper left panel) the channel was open
less than 0.1%. Only a 2-minute control segment is shown, but this behavior was maintained for 10 minutes. Addition of 2.5 mM caffeine at the beginning of the segment shown led to increased channel activity (Figure 4, second plot of upper left panel). The third and fourth plots show channel activity 10 and 20 minutes after caffeine addition. Although the mean open time varied between 52% and 29% in this experiment, it does not indicate that the activity is decreasing with time. Variation in the mean activity level as seen in this figure was found in virtually all experiments performed, regardless of the concentration of caffeine used. This variability appears to be a property of the calcium release channel from SR. For example, two additional experiments are shown in Figure 5, in which the degree of activation varied between 18% and 82%. The duration of activation was similar after addition of either 2.5 or 5 mM caffeine (Figure 5). In 10 experiments in which caffeine was the activator, the channels were active for a period greater than 15 minutes (16-90 minutes; mean, 44 minutes). A second addition of caffeine reactivated the channel.
Control
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FIGURE 2. Recordings showing acute effects of caffeine and doxorubicin on single-channel currents of cardiac calcium release channels. Single-channel recordings in control conditions (0.1 pum free calcium) had a open probability of 200 times less intense in the ultraviolet range). In summary, we have shown that both caffeine and doxorubicin can activate the calcium release channel from cardiac SR but that doxorubicin induced inhibition of the channel after prolonged exposure. Dox-
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orubicin-induced inactivation of the channel was prevented by pretreating the system with DPI?. These experiments show that activation and inactivation of the calcium release channel can be separated, suggesting that different biochemical mechanisms are involved in the two responses to doxorubicin addition. These experiments also show that the biphasic effects of doxorubicin on the calcium release channel can be related to the observed cardiotoxicity. Acknowledgments We gratefully acknowledge Dr. James Watras for assistance in data analysis and Drs. Arnold Katz and James Watras, Department of Medicine, University of Connecticut, Farmington, for helpful comments on the manuscript. References 1. Imagawa T, Smith, JS, Coronado R, Campell KP: Purified ryanodine receptor from skeletal muscle sarcoplasmic reticulum is the calcium permeable pore of the calcium release
channel. J Biol Chem 1987;262:16636-16643 2. Inui M, Saito A, Fleischer S: Isolation of the ryanodine receptor from cardiac sarcoplasmic reticulum and identity with the feet structures. J Biol Chem 1987;262:15637-15642 3. Lai FA, Erickson HP, Rousseau E, Liu Q-Y, Meissner G: Purification and reconstitution of the calcium release channel from skeletal muscle. Nature 1988;331:315-319 4. Takeshima H, Nishimura S, Matsumoto T, Ishida H, Kangawa K, Minamino N, Matsuo H, Ueda M, Hanaoka M, Hirose T, Numa S: Primary structure and expression from complementary DNA of skeletal muscle ryanodine receptor. Nature 1989;339:439-445 5. Smith J, Coronado R, Meissner G: Single channel measurements of the calcium release channel from skeletal muscle sarcoplasmic reticulum: Activation by calcium and ATP and modulation by magnesium. J Gen Physiol 1986;88:573-588 6. Rousseau E, Smith J, Henderson J, Meissner G: Single channel and calcium flux measurements of the cardiac sarcoplasmic reticulum calcium channel. Biophys J 1986;50: 1009-1014 7. Ehrlich BE, Watras J: Inositol 1,4,5-trisphosphate activates a channel from smooth muscle sarcoplasmic reticulum. Nature 1988;336:583-586 8. Tanabe T, Beam KG, Powell JA, Numa S: Restoration of excitation-contraction coupling and slow calcium current in dysgenic muscle by dihydropyridine receptor complementary DNA. Nature 1988;336:134-149 9. Fabiato A, Fabiato F: Use of chlorotetracycline fluorescence to demonstrate calcium-induced release of calcium from the
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15. Abramson JJ, Buck E, Salama G, Casida JE, Pessah IN: Mechanism of anthraquinone-induced calcium release from skeletal muscle sarcoplasmic reticulum. J Biol Chem 1988;263: 18750-18758 16. Powis G: Free radical formation by antitumor quinones. Free Radic Biol Med 1989;6:63-101 17. Singal PK: Subcellular effects of adriamycin in the heart: A concise review. J Mol Cell Cardiol 1987;9:817-828 18. Zorzato F, Salviati G, Facchinetti T, Volpe P: Doxorubicin induces calcium release from terminal cisternae of skeletal muscle. J Biol Chem 1985;260:7349-7355 19. Kranias EG, Schwartz A, Jungmann RA: Characterization of cyclic 3'5 '-AMP-dependent protein kinase in sarcoplasmic reticulum and cytosol of canine myocardium. Biochim Biophys Acta 1982;709:28-37 20. Kim DH, Ikemoto N: Involvement of 60-kilodalton phosphoprotein in the regulation of calcium release from skeletal muscle sarcoplasmic reticulum. J Biol Chem 1986;261: 11674-11679 21. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265-275 22. Kim HW, Kim DH, Ikemoto N, Kranias EG: Lack of effect of calcium calmodulin-dependent phosphorylation on Ca2' release from cardiac sarcoplasmic reticulum. Biochim Biophys Acta 1987;903:333-340 23. Mueller P, Rudin DO, Tien HT, Wescott WC: Methods for the incorporation of single bimolecular lipid membranes in aqueous solutions. J Phys Chem 1963;67:534-535 24. Watras J, Benevolensky D: Inositol 1,4,5, triphosphate-induced calcium release from canine aortic sarcoplasmic reticulum vesicles. Biochim Biophys Acta 1987;931:354-363 25. Kim DH, Ohnishi ST, Ikemoto N: Kinetic studies of calcium release from sarcoplasmic reticulum in vitro. J Biol Chem 1983;258:9662-9668
26. Benevolensky DS, Menshikova EV, Watras J, Levitsky DO, Ritov VB: Characterization of Ca2+ release from the sarcoplasmic reticulum of myocardium and vascular smooth muscle. Biomed Biochim Acta 1987;46:S393-S398 27. Rousseau E, LaDine J, Liu Q-Y, Meissner G: Activation of the calcium release channel of the skeletal muscle sarcoplasmic reticulum by caffeine and related compounds. Arch Biochem Biophys 1988;267:75-86 28. Rousseau E, Meissner G: Single cardiac sarcoplasmic reticulum Ca2+-release channel: Activation by caffeine. Am J Physiol 1989;256:H328-H333 29. Preisler HD, Gessner T, Azarnia N, Bolanowska W, Epstein J, Early AP, D'Arrigo P, Vogler R, Winton L, Chervenik P, Joyce R, Lee H, Steele R, Goldberg J, Gottlieb A, Browman G, Miller K, Grunwald H, Larson R, Brennan J: Relationship between plasma adriamycin levels and the outcome of remission induction therapy for acute nonlymphocytic leukemia. Cancer Chemother Pharmacol 1984;12:125-130 30. Cortes EP, Gupta M, Chou C, Amin VC, Folkers K: Adriamycin cardiotoxicity: Early detection by systolic time interval and possible prevention by coenzyme Q10. Cancer Treat Reports 1978;62:887-891 31. Harris RN, Doroshow JH: Effect of doxorubicin-enhanced hydrogen peroxide and hydroxyl radical formation on calcium sequestration by cardiac sarcoplasmic reticulum. Biochem Biophys Res Commun 1985;130:739-745 32. Singal PK, Pierce GN: Adriamycin stimulates low-affinity Ca2' binding and lipid peroxidation but depresses myocardial function. Am J Physiol 1986;250:H419-H425
KEY WORDS * sarcoplasmic reticulum * planar lipid bilayers doxorubicin cardiotoxicity * calcium channels * single ion channels
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Biphasic effects of doxorubicin on the calcium release channel from sarcoplasmic reticulum of cardiac muscle. K Ondrias, L Borgatta, D H Kim and B E Ehrlich Circ Res. 1990;67:1167-1174 doi: 10.1161/01.RES.67.5.1167 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1990 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571
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